Maleate salts of a quinazoline derivative useful as an antiangiogenic agent

ABSTRACT

The present invention relates to AZD2171 maleate salt, to particular crystalline forms of AZD2171 maleate salt, to processes for their preparation, to pharmaceutical compositions containing them as active ingredient, to their use in the manufacture of medicaments for use in the production of antiangiogenic and/or vascular permeability reducing effects in warm-blooded animals such as humans, and to their use in methods for the treatment of disease states associated with angiogenesis and/or increased vascular permeability.

The present invention relates to AZD2171 maleate salt, to particularcrystalline forms of AZD2171 maleate salt, to processes for theirpreparation, to pharmaceutical compositions containing them as activeingredient, to their use in the manufacture of medicaments for use inthe production of antiangiogenic and/or vascular permeability reducingeffects in warm-blooded animals such as humans, and to their use inmethods for the treatment of disease states associated with angiogenesisand/or increased vascular permeability.

Normal angiogenesis plays an important role in a variety of processesincluding embryonic development, wound healing and several components offemale reproductive function. Undesirable or pathological angiogenesishas been associated with disease states including diabetic retinopathy,psoriasis, cancer, rheumatoid arthritis, atheroma, Kaposi's sarcoma andhaemangioma (Fan et al, 1995, Trends Pharmacol. Sci. 16: 57-66; Folkman,1995, Nature Medicine 1: 27-31). Alteration of vascular permeability isthought to play a role in both normal and pathological physiologicalprocesses (Cullinan-Bove et al, 1993, Endocrinology 133: 829-837; Sengeret al, 1993, Cancer and Metastasis Reviews, 12: 303-324). Severalpolypeptides with in vitro endothelial cell growth promoting activityhave been identified including, acidic and basic fibroblast growthfactors (aFGF & bFGF) and vascular endothelial growth factor (VEGF). Byvirtue of the restricted expression of its receptors, the growth factoractivity of VEGF, in contrast to that of the FGFs, is relativelyspecific towards endothelial cells. Recent evidence indicates that VEGFis an important stimulator of both normal and pathological angiogenesis(Jakeman et al, 1993, Endocrinology, 133: 848-859; Kolch et al, 1995,Breast Cancer Research and Treatment, 36:139-155) and vascularpermeability (Connolly et al, 1989, J. Biol. Chem. 264: 20017-20024).Antagonism of VEGF action by sequestration of VEGF with antibody canresult in inhibition of tumour growth (Kim et al, 1993, Nature 362:841-844).

Receptor tyrosine kinases (RTKs) are important in the transmission ofbiochemical signals across the plasma membrane of cells. Thesetransmembrane molecules characteristically consist of an extracellularligand-binding domain connected through a segment in the plasma membraneto an intracellular tyrosine kinase domain. Binding of ligand to thereceptor results in stimulation of the receptor-associated tyrosinekinase activity which leads to phosphorylation of tyrosine residues onboth the receptor and other intracellular molecules. These changes intyrosine phosphorylation initiate a signalling cascade leading to avariety of cellular responses. To date, at least nineteen distinct RTKsubfamilies, defined by amino acid sequence homology, have beenidentified. One of these subfamilies is presently comprised by thefms-like tyrosine kinase receptor, Flt-1, the kinase insertdomain-containing receptor, KDR (also referred to as Flk-1), and anotherfms-like tyrosine kinase receptor, Flt-4. Two of these related RTKs,Flt-1 and KDR, have been shown to bind VEGF with high affinity (De Vrieset al, 1992, Science 255: 989-991; Terman et al, 1992, Biochem. Biophys.Res. Comm. 1992, 187: 1579-1586). Binding of VEGF to these receptorsexpressed in heterologous cells has been associated with changes in thetyrosine phosphorylation status of cellular proteins and calcium fluxes.

VEGF is a key stimulus for vasculogenesis and angiogenesis. Thiscytokine induces a vascular sprouting phenotype by inducing endothelialcell proliferation, protease expression and migration, and subsequentorganisation of cells to form a capillary tube (Keck, P. J., Hauser, S.D., Krivi, G., Sanzo, K., Warren, T., Feder, J., and Connolly, D. T.,Science (Washington D.C.), 246: 1309-1312, 1989; Lamoreaux, W. J.,Fitzgerald, M. E., Reiner, A., Hasty, K. A., and Charles, S. T.,Microvasc. Res., 55: 29-42, 1998; Pepper, M. S., Montesano, R.,Mandroita, S. J., Orci, L. and Vassalli, J. D., Enzyme Protein, 49:138-162, 1996.). In addition, VEGF induces significant vascularpermeability (Dvorak, H. F., Detmar, M., Claffey, K. P., Nagy, J. A.,van de Water, L., and Senger, D. R., (Int. Arch. Allergy Immunol., 107:233-235, 1995; Bates, D. O., Heald, R. I., Curry, F. E. and Williams, B.J. Physiol. (Lond.), 533: 263-272, 2001), promoting formation of ahyper-permeable, immature vascular network which is characteristic ofpathological angiogenesis.

It has been shown that activation of KDR alone is sufficient to promoteall of the major phenotypic responses to VEGF, including endothelialcell proliferation, migration, and survival, and the induction ofvascular permeability (Meyer, M., Clauss, M., Lepple-Wienhues, A.,Waltenberger, J., Augustin, H. G., Ziche, M., Lanz, C., Buttner, M.,Rziha, H-J., and Dehio, C., EMBO J., 18: 363-374, 1999; Zeng, H.,Sanyal, S. and Mukhopadhyay, D., J. Biol. Chem., 276: 32714-32719, 2001;Gille, H., Kowalski, J., Li, B., LeCouter, J., Moffat, B, Zioncheck, T.F., Pelletier, N. and Ferrara, N., J. Biol. Chem., 276: 3222-3230,2001).

Compounds which inhibit the effects of VEGF are of value in thetreatment of disease states associated with angiogenesis and/orincreased vascular permeability such as cancer (including leukaemia,multiple myeloma and lymphoma), diabetes, psoriasis, rheumatoidarthritis, Kaposi's sarcoma, haemangioma, acute and chronicnephropathies, atheroma, arterial restenosis, autoimmune diseases, acuteinflammation, excessive scar formation and adhesions, endometriosis,lymphoedema, dysfunctional uterine bleeding and ocular diseases withretinal vessel proliferation including macular degeneration.

Quinazoline derivatives which are inhibitors of VEGF receptor tyrosinekinase are described in WO 00/47212. The compound AZD2171 is exemplifedin WO 00/47212, (see Example 240), and is4-((4-fluoro-2-methyl-1H-indol-5-yl)oxy)-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazolineof the formula I:

AZD2171 shows excellent activity in the in vitro (a) enzyme and (b)HUVEC assays that are described in WO 00/47212 and hereinafter. TheAZD2171 IC₅₀ values for inhibition of isolated KDR (VEGFR-2) and Flt-1(VEGFR-1) tyrosine kinase activities in the enzyme assay were <2 nM and5±2 nM respectively. AZD2171 inhibits VEGF-stimulated endothelial cellproliferation potently (IC₅₀ value of 0.4±0.2 nM in the HUVEC assay),but does not inhibit basal endothelial cell proliferation appreciably ata >1250 fold greater concentration (IC₅₀ value is >500 nM). The growthof a Calu-6 tumour xenograft in the in vivo (c) solid tumour modeldescribed hereinafter was inhibited by 49%**, 69%*** and 91%***following 28 days of once-daily oral treatment with 1.5, 3 and 6mg/kg/day AZD2171 respectively (P**<0.01, P***<0.0001; one-tailed ttest).

More stable forms of a pharmaceutically active compound, for examplemore stable crystalline forms, are preferred for formulation andprocessing on a commercial scale. This is because the greater thestability of the form used, the lower the risk of it converting toanother form during formulation procedures such as compression. This inturn provides greater predictability of the properties of the finalformulation, such as dissolution rate of tablets, bioavailability ofactive ingredient. Using a more stable form of an active ingredientallows greater control over the physical properties of the formulation.

AZD2171 free base(4-((4-fluoro-2-methyl-1H-indol-5-yl)oxy)-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazoline)is a crystalline monohydrate under ambient conditions. DifferentialScanning calorimetry (DSC) analysis was carried out according to themethod described hereinafter and shows a large broad endotherm between95° and 170° C. due to loss of water and melting (FIG. 1).Thermogravimetric (TGA) analysis (details given hereinafter) shows aweight loss of 4.02% between 80° C. and 115° C. (FIG. 1). Karl Fischerwater analysis (details given hereinafter) yields a figure of 3.9%suggesting that all the weight loss is due to water loss.

It will be understood that the onset/peak temperature values of the DSCmay vary slightly from one machine to another or from one sample toanother, and so the values quoted are not to be construed as absolute.

AZD2171 free base is characterised in providing at least one of thefollowing 20 values measured using CuKa radiation: 18.3 and 20.8.AZD2171 free base is characterised in providing an X-ray powderdiffraction pattern, as in FIG. 2. The ten most prominent peaks areshown in Table 1.

TABLE 1 Ten most Prominent X-Ray Powder Diffraction peaks for AZD2171free base Angle 2- Intensity Relative Theta (2θ) Count Intensity 18.287100 vs 20.807 66.7 vs 27.277 48.9 vs 23.370 42.8 vs 14.684 39.8 vs25.070 37.6 vs 13.966 32.2 vs 21.711 26.6 vs 22.898 23.1 vs 26.790 22.9vs vs = very strong

It has been found that when a sample of AZD2171 free base is dehydrated,for example on heating to 100° C., the sample becomes amorphous (FIG. 3)and does not then rehydrate but stays amorphous thereafter. This couldbe problematic if AZD2171 free base were to be formulated as apharmaceutical composition because AZD2171 free base could dehydrateduring certain processes e.g. particle size reduction (such as milling),drying of bulk drug, formulating, manufacturing. In order to formulateAZD2171 free base as a pharmaceutical composition it would be necessaryto reduce the particle size at some point, and this would carry a riskof dehydration and therefore the risk of the formation of amorphousmaterial. This was investigated by subjecting a sample of AZD2171 freebase monohydrate to particle size reduction by micronisation and thenanalysing it to look for amorphous material. FIG. 4 shows that amorphousmaterial does indeed form during particle size reduction of AZD2171 freebase. This is shown by a broadening of the peaks and formation of anamorphous ‘hump’—see FIG. 4. An amorphous or semi-amorphous form ofAZD2171 free base could give rise to manufacturing problems andnon-reproducible bioavailability.

The identification of alternative forms of AZD2171, forms that aredifferent from the free base and that have improved solid stateproperties, is the subject of the present invention.

An example of a different form is a salt of AZD2171. In WO 00/47212 itsays that pharmaceutically acceptable salts of the compounds of theinvention therein may include acid addition salts of the compounds ofthe invention which are sufficiently basic to form such salts. Such acidaddition salts are said to include salts with inorganic or organic acidsaffording pharmaceutically acceptable anions such as with hydrogenhalides especially hydrochloric or hydrobromic acid or with sulphuric orphosphoric acid, or with trifluoroacetic, citric or maleic acid. Inaddition WO 00/47212 goes on to say that where the compounds of theinvention therein are sufficiently acidic, pharmaceutically acceptablesalts may be formed with an inorganic or organic base which affords apharmaceutically acceptable cation. Such salts with inorganic or organicbases are said to include an alkali metal salt, such as a sodium orpotassium salt, an alkaline earth metal salt such as a calcium ormagnesium salt, an ammonium salt or for example a salt with methylamine,dimethylamine, trimethylamine, piperidine, morpholine ortris-(2-hydroxyethyl)amine.

Preferred salts in WO 00/47212 are hydrochlorides and hydrobromides,especially hydrochlorides.

Nowhere in WO 00/47212 does it state that a particular salt of aparticular compound therein will possess surprisingly beneficialproperties.

Unexpectedly and surprisingly we have now found that the maleate salt ofAZD2171 is an advantageously stable form of AZD2171 with improved solidstate properties over the free base and over other salts that have beentested.

AZD2171 maleate is readily crystallised, is highly crystalline,non-hygroscopic and has a reproducible stoichiometric ratio of drug tocounter-ion of 1:1.

Thus AZD2171 maleate is readily crystallised, is highly crystalline,non-hygroscopic and has a reproducible stoichiometric ratio of drug tocounter-ion of about 1:1.

Several salts of AZD2171 were prepared and seven were found to becrystalline: malonate, succinate, fumarate, maleate, tartarate, adipateand malate. The solid state properties of these 7 salts were tested andthe results are shown in Table 2:

TABLE 2 Properties of AZD2171 Salts Evidence Moisture of HydrateCrystalline Drug:Counter-ion Content at Formation^(b) No of Salt(Yes/No) Stoichiometry^(a) 80% RH^(b) (Yes/No) Polymorphs^(c) MalonateYes — — Yes ≥3 Succinate Yes 1:0.63 11.4  No ≥2 Fumarate Yes 1:0.5  3.5No ≥3 Maleate Yes 1:1   0.4 No ≥2 Tartarate Yes 1:0.75 9.3 No ≥1 AdipateYes 1:0.75 — No ≥3 Malate Yes — 7.7 Yes — ^(a)Drug:counterionstoichiometry from ¹H NMR Spectrum data ^(b)Moisture content at 80%relative humidity (RH). Evidence of hydration from Vapour Sorptionstudies (observed hysteresis and absorption of water) orThermogravimetric Analysis (TGA) ^(c)Evidence for polymorphism fromDifferential Scanning Calorimetry (DSC) thermogramsThe term ‘non-hygroscopic’ means absorbing <1% moisture at 80% RH.

The AZD2171 maleate salt was surprisingly better than the others becauseof the 7 salts that it was possible to crystallise, it was found to bethe only non-hygroscopic salt, to be highly crystalline and to have areproducible stoichiometric ratio of drug to counter-ion of 1:1.

Thus AZD2171 maleate was found to be the only non-hygroscopic salt, tobe highly crystalline and to have a reproducible stoichiometric ratio ofdrug to counter-ion of about 1:1.

AZD2171 maleate salt is substantially free of amorphous material and canbe expected to be easier to formulate than AZD2171 free base and toprovide more reproducible dosing results. By “substantially free ofamorphous material” is meant that the amount of amorphous material isless than 10%, preferably less than 5%, more preferably less than 2%.

AZD2171 maleate salt is non-hygroscopic which should prevent or reduceany problems associated with weight changes of the active ingredientduring procedures such as micronisation.

According to the present invention there is provided a maleate salt ofAZD2171.

AZD2171 maleate has two crystalline forms A and B.

According to the present invention there is provided a maleate salt ofAZD2171 in a first crystalline form, Form A.

AZD2171 Maleate Form A is characterised in providing at least one of thefollowing 28 values measured using CuKa radiation: 21.5 and 16.4.AZD2171 Maleate Form A is characterised in providing an X-ray powderdiffraction pattern, substantially as shown in FIG. 5. The ten mostprominent peaks are shown in Table 3:

TABLE 3 Ten most Prominent X-Ray Powder Diffraction peaks for AZD2171Maleate Form A Angle 2- Intensity Relative Theta (2θ) Count Intensity21.522 100 vs 16.366 78.3 vs 24.381 73.7 vs 20.721 71.7 vs 25.025 71.5vs 16.921 55.5 vs 12.085 44.1 vs 22.177 42.2 vs 17.444 40.7 vs 17.62739.1 vs vs = very strong

According to the present invention there is provided a maleate salt ofAZD2171 in a first crystalline form, Form A, wherein said salt has anX-ray powder diffraction pattern with at least one specific peak atabout 2-theta=21.5°.

According to the present invention there is provided a maleate salt ofAZD2171 in a first crystalline form, Form A, wherein said salt has anX-ray powder diffraction pattern with at least one specific peak atabout 2-theta=16.4°.

According to the present invention there is provided a maleate salt ofAZD2171 in a first crystalline form, Form A, wherein said salt has anX-ray powder diffraction pattern with at least two specific peaks atabout 2-theta=21.5° and 16.4°.

According to the present invention there is provided a maleate salt ofAZD2171 in a first crystalline form, Form A, wherein said salt has anX-ray powder diffraction pattern with specific peaks at about2-theta=21.5, 16.4, 24.4, 20.7, 25.0, 16.9, 12.1, 22.2, 17.4 and 17.6°.

According to the present invention there is provided a maleate salt ofAZD2171 in a first crystalline form, Form A, wherein said salt has anX-ray powder diffraction pattern substantially the same as the X-raypowder diffraction pattern shown in FIG. 5.

According to the present invention there is provided a maleate salt ofAZD2171 in a first crystalline form, Form A, wherein said salt has anX-ray powder diffraction pattern with at least one specific peak at2-theta=21.5° plus or minus 0.5° 2-theta.

According to the present invention there is provided a maleate salt ofAZD2171 in a first crystalline form, Form A, wherein said salt has anX-ray powder diffraction pattern with at least one specific peak at2-theta=16.4° plus or minus 0.5° 2-theta.

According to the present invention there is provided a maleate salt ofAZD2171 in a first crystalline form, Form A, wherein said salt has anX-ray powder diffraction pattern with at least two specific peaks at2-theta=21.5° and 16.4° wherein said values may be plus or minus 0.5°2-theta.

According to the present invention there is provided a maleate salt ofAZD2171 in a first crystalline form, Form A, wherein said salt has anX-ray powder diffraction pattern with specific peaks at 2-theta=21.5,16.4, 24.4, 20.7, 25.0, 16.9, 12.1, 22.2, 17.4 and 17.6° wherein saidvalues may be plus or minus 0.5° 2-theta.

According to the present invention there is provided a maleate salt ofAZD2171 in a first crystalline form, Form A, wherein said salt has anX-ray powder diffraction pattern with at least one specific peak at2-theta=21.5°.

According to the present invention there is provided a maleate salt ofAZD2171 in a first crystalline form, Form A, wherein said salt has anX-ray powder diffraction pattern with at least one specific peak at2-theta=16.4°.

According to the present invention there is provided a maleate salt ofAZD2171 in a first crystalline form, Form A, wherein said salt has anX-ray powder diffraction pattern with at least two specific peaks at2-theta=21.5° and 16.4°.

According to the present invention there is provided a maleate salt ofAZD2171 in a first crystalline form, Form A, wherein said salt has anX-ray powder diffraction pattern with specific peaks at 2-theta=21.5,16.4, 24.4, 20.7, 25.0, 16.9, 12.1, 22.2, 17.4 and 17.6°.

According to the present invention there is provided a maleate salt ofAZD2171 in a first crystalline form, Form A, wherein said salt has anX-ray powder diffraction pattern as shown in FIG. 5.

DSC analysis shows AZD2171 maleate Form A is a high melting solid withan onset of melting at 198.3° C. and a peak at 200.08° C. (FIG. 6).

Thus DSC analysis shows AZD2171 maleate Form A is a high melting solidwith an onset of melting at about 198.3° C. and a peak at about 200.08°C.

According to the present invention there is provided a maleate salt ofAZD2171 in a second crystalline form, Form B.

AZD2171 Maleate Form B is characterised in providing at least one of thefollowing 28 values measured using CuKa radiation: 24.2 and 22.7.AZD2171 Maleate Form B is characterised in providing an X-ray powderdiffraction pattern substantially as shown in FIG. 8. The ten mostprominent peaks are shown in Table 4:

TABLE 4 Ten most Prominent X-Ray Powder Diffraction peaks for AZD2171Maleate Form B Angle 2- Intensity Relative Theta (2θ) Count Intensity24.156 100 vs 22.740 84.3 vs 15.705 64.0 vs 11.995 63.7 vs 27.087 60.9vs 25.032 56.8 vs 17.724 37.7 vs 15.044 35.4 vs 23.102 34.5 vs 12.62534.2 vs vs = very strong

According to the present invention there is provided a maleate salt ofAZD2171 in a second crystalline form, Form B, wherein said salt has anX-ray powder diffraction pattern with at least one specific peak atabout 2-theta=24.2°.

According to the present invention there is provided a maleate salt ofAZD2171 in a second crystalline form, Form B, wherein said salt has anX-ray powder diffraction pattern with at least one specific peak atabout 2-theta=22.7°.

According to the present invention there is provided a maleate salt ofAZD2171 in a second crystalline form, Form B, wherein said salt has anX-ray powder diffraction pattern with at least two specific peaks atabout 2-theta=24.2° and 22.7°.

According to the present invention there is provided a maleate salt ofAZD2171 in a second crystalline form, Form B, wherein said salt has anX-ray powder diffraction pattern with specific peaks at about2-theta=24.2, 22.7, 15.7, 12.0, 27.1, 25.0, 17.7, 15.0, 23.1 and 12.6°.

According to the present invention there is provided a maleate salt ofAZD2171 in a second crystalline form, Form B, wherein said salt has anX-ray powder diffraction pattern substantially the same as the X-raypowder diffraction pattern shown in FIG. 8.

According to the present invention there is provided a maleate salt ofAZD2171 in a second crystalline form, Form B, wherein said salt has anX-ray powder diffraction pattern with at least one specific peak at2-theta=24.2° plus or minus 0.5° 2-theta.

According to the present invention there is provided a maleate salt ofAZD2171 in a second crystalline form, Form B, wherein said salt has anX-ray powder diffraction pattern with at least one specific peak at2-theta=22.7° plus or minus 0.5° 2-theta.

According to the present invention there is provided a maleate salt ofAZD2171 in a second crystalline form, Form B, wherein said salt has anX-ray powder diffraction pattern with at least two specific peaks at2-theta=24.2° and 22.7° wherein said values may be plus or minus 0.5°2-theta.

According to the present invention there is provided a maleate salt ofAZD2171 in a second crystalline form, Form B, wherein said salt has anX-ray powder diffraction pattern with specific peaks at 2-theta=24.2,22.7, 15.7, 12.0, 27.1, 25.0, 17.7, 15.0, 23.1 and 12.6° wherein saidvalues may be plus or minus 0.5° 2-theta.

According to the present invention there is provided a maleate salt ofAZD2171 in a second crystalline form, Form B, wherein said salt has anX-ray powder diffraction pattern with at least one specific peak at2-theta=24.2°.

According to the present invention there is provided a maleate salt ofAZD2171 in a second crystalline form, Form B, wherein said salt has anX-ray powder diffraction pattern with at least one specific peak at2-theta=22.7°.

According to the present invention there is provided a maleate salt ofAZD2171 in a second crystalline form, Form B, wherein said salt has anX-ray powder diffraction pattern with at least two specific peaks at2-theta=24.2° and 22.7°.

According to the present invention there is provided a maleate salt ofAZD2171 in a second crystalline form, Form B, wherein said salt has anX-ray powder diffraction pattern with specific peaks at 2-theta=24.2,22.7, 15.7, 12.0, 27.1, 25.0, 17.7, 15.0, 23.1 and 12.6°.

According to the present invention there is provided a maleate salt ofAZD2171 in a second crystalline form, Form B, wherein said salt has anX-ray powder diffraction pattern as shown in FIG. 8.

DSC analysis shows AZD2171 maleate Form B is a high melting solid withan onset of melting at 194.43° C. and a peak at 195.97° C. (FIG. 9).

Thus DSC analysis shows AZD2171 maleate Form B is a high melting solidwith an onset of melting at about 194.43° C. and a peak at about 195.97°C.

Form B is meta-stable with respect to Form A (the melting point and heatof fusion of Form B are lower than those for Form A). Form A is the morethermodynamically stable form. A mixture of Form A and B converts toForm A upon slurrying in methanol at 40° C. for 4 days (FIG. 10).

Form A is preferred over Form B.

AZD2171 maleate is non-hygroscopic, absorbing <1% moisture at 80%relative humidity (FIG. 7).

The NMR details are given after the maleate salt preparation in Example1 and show for the stoichiometry data a ratio of 1:1.

According to another aspect of the present invention there is provided adimaleate salt of AZD2171. A dimaleate salt my be formed through theaddition of two moles of maleic acid to one mole of AZD2171 free base.

When it is stated that the present invention relates to a crystallineform of AZD2171 free base, or AZD2171 maleate Form A or AZD2171 maleateForm B, the degree of crystallinity is conveniently greater than about60%, more conveniently greater than about 80%, preferably greater thanabout 90% and more preferably greater than about 95%. Most preferablythe degree of crystallinity is greater than about 98%.

The AZD2171 maleate salt forms A and B provide X-ray powder diffractionpatterns substantially the same as the X-ray powder diffraction patternsshown in FIGS. 5 and 8 respectively and have substantially the ten mostprominent peaks (angle 2-theta values) shown in Tables 3 and 4respectively. It will be understood that the 2-theta values of the X-raypowder diffraction pattern may vary slightly from one machine to anotheror from one sample to another, and so the values quoted are not to beconstrued as absolute.

It is known that an X-ray powder diffraction pattern may be obtainedwhich has one or more measurement errors depending on measurementconditions (such as equipment or machine used). In particular, it isgenerally known that intensities in an X-ray powder diffraction patternmay fluctuate depending on measurement conditions. Therefore it shouldbe understood that the AZD2171 maleate salt forms of the presentinvention are not limited to the crystals that provide X-ray powderdiffraction patterns identical to the X-ray powder diffraction patternsshown in FIGS. 5 and 8, and any crystals providing X-ray powderdiffraction patterns substantially the same as those shown in FIGS. 5and 8 fall within the scope of the present invention. A person skilledin the art of X-ray powder diffraction is able to judge the substantialidentity of X-ray powder diffraction patterns.

Persons skilled in the art of X-ray powder diffraction will realise thatthe relative intensity of peaks can be affected by, for example, grainsabove 30 microns in size and non-unitary aspect ratios, which may affectanalysis of samples. The skilled person will also realise that theposition of reflections can be affected by the precise height at whichthe sample sits in the diffractometer and the zero calibration of thediffractometer. The surface planarity of the sample may also have asmall effect. Hence the diffraction pattern data presented are not to betaken as absolute values. (Jenkins, R & Snyder, R. L. ‘Introduction toX-Ray Powder Diffractometry’ John Wiley & Sons 1996; Bunn, C. W. (1948),Chemical Crystallography, Clarendon Press, London; Klug, H. P. &Alexander, L. E. (1974), X-Ray Diffraction Procedures).

Generally, a measurement error of a diffraction angle in an X-ray powderdiffractogram is about 5% or less, in particular plus or minus 0.5°2-theta, and such degree of a measurement error should be taken intoaccount when considering the X-ray powder diffraction patterns in FIGS.2, 3, 4, 5, 8 and 10 and when reading Tables 1, 3 and 4. Furthermore, itshould be understood that intensities may fluctuate depending onexperimental conditions and sample preparation (preferred orientation).

For the avoidance of doubt, terms such as ‘AZD2171 maleate salt’ and ‘amaleate salt of AZD2171’ refer to each and every form of AZD2171 maleatesalt, whereas ‘AZD2171 maleate Form A’ refers to the particularcrystalline form known as Form A and ‘AZD2171 maleate Form B’ refers tothe particular crystalline form known as Form B.

According to a further aspect of the invention there is provided apharmaceutical composition which comprises an AZD2171 maleate salt asdefined hereinbefore in association with a pharmaceutically acceptableexcipient or carrier.

The composition may be in a form suitable for oral administration, (forexample as tablets, lozenges, hard or soft capsules, aqueous or oilysuspensions, emulsions, dispersible powders or granules, syrups orelixirs), for administration by inhalation (for example as a finelydivided powder or a liquid aerosol), for administration by insufflation(for example as a finely divided powder), for parenteral injection (forexample as a sterile solution, suspension or emulsion for intravenous,subcutaneous, intramuscular, intravascular or infusion dosing), fortopical administration (for example as creams, ointments, gels, oraqueous or oily solutions or suspensions), or for rectal administration(for example as a suppository). Preferably AZD2171 maleate salt isadministered orally. In general the above compositions may be preparedin a conventional manner using conventional excipients.

The compositions of the present invention are advantageously presentedin unit dosage form. AZD2171 maleate will normally be administered to awarm-blooded animal at a unit dose within the range 1-50 mg per squaremetre body area of the animal, for example approximately 0.03-1.5 mg/kgin a human. A unit dose in the range, for example, 0.01-1.5 mg/kg, forexample 0.05-0.75 mg/kg, preferably 0.03-0.5 mg/kg is envisaged and thisis normally a therapeutically-effective dose. A unit dosage form such asa tablet or capsule will usually contain, for example 1-50 mg of activeingredient. Preferably a daily dose in the range of 0.03-0.5 mg/kg isemployed. The size of the dose required for the therapeutic orprophylactic treatment of a particular disease state will necessarily bevaried depending on the host treated, the route of administration andthe severity of the illness being treated. Accordingly the optimumdosage may be determined by the practitioner who is treating anyparticular patient.

According to a further aspect of the present invention there is providedan AZD2171 maleate salt as defined hereinbefore for use in a method oftreatment of the human or animal body by therapy.

A further feature of the present invention is an AZD2171 maleate salt asdefined hereinbefore for use as a medicament, conveniently an AZD2171maleate salt as defined hereinbefore for use as a medicament forproducing an antiangiogenic and/or vascular permeability reducing effectin a warm-blooded animal such as a human being.

Thus according to a further aspect of the invention there is providedthe use of an AZD2171 maleate salt as defined hereinbefore in themanufacture of a medicament for use in the production of anantiangiogenic and/or vascular permeability reducing effect in awarm-blooded animal such as a human being.

According to a further feature of the invention there is provided amethod for producing an antiangiogenic and/or vascular permeabilityreducing effect in a warm-blooded animal, such as a human being, in needof such treatment which comprises administering to said animal aneffective amount of an AZD2171 maleate salt as defined hereinbefore.

AZD2171 maleate salt is an antiangiogenic and/or vascular permeabilityreducing agent and may be applied as a sole therapy or may involve, inaddition to AZD2171 maleate, one or more other substances and/ortreatments. Such conjoint treatment may be achieved by way of thesimultaneous, sequential or separate administration of the individualcomponents of the treatment. In the field of medical oncology it isnormal practice to use a combination of different forms of treatment totreat each patient with cancer. In medical oncology the othercomponent(s) of such conjoint treatment in addition to AZD2171 maleatesalt may be: surgery, radiotherapy or chemotherapy. Such chemotherapymay cover three main categories of therapeutic agent:

(i) other antiangiogenic agents such as those which inhibit the effectsof vascular endothelial growth factor, (for example the anti-vascularendothelial cell growth factor antibody bevacizumab [Avastin™], andthose that work by different mechanisms from those defined hereinbefore(for example linomide, inhibitors of integrin αvβ3 function,angiostatin, razoxin, thalidomide), and including vascular targetingagents (for example combretastatin phosphate and compounds disclosed inInternational Patent Applications WO00/40529, WO 00/41669, WO01/92224,WO02/04434 and WO02/08213 and the vascular damaging agents described inInternational Patent Application Publication No. WO 99/02166 the entiredisclosure of which document is incorporated herein by reference, (forexample N-acetylcolchinol-O-phosphate));(ii) cytostatic agents such as antioestrogens (for example tamoxifen,toremifene, raloxifene, droloxifene, iodoxyfene), oestrogen receptordown regulators (for example fulvestrant), progestogens (for examplemegestrol acetate), aromatase inhibitors (for example anastrozole,letrazole, vorazole, exemestane), antiprogestogens, antiandrogens (forexample flutamide, nilutamide, bicalutamide, cyproterone acetate), LHRHagonists and antagonists (for example goserelin acetate, luprolide,buserelin), inhibitors of 5α-reductase (for example finasteride),anti-invasion agents (for example metalloproteinase inhibitors likemarimastat and inhibitors of urokinase plasminogen activator receptorfunction) and inhibitors of growth factor function, (such growth factorsinclude for example platelet derived growth factor and hepatocyte growthfactor), such inhibitors include growth factor antibodies, growth factorreceptor antibodies, (for example the anti-erbb2 antibody trastuzumab[Herceptin™] and the anti-erbb1 antibody cetuximab [C225]), farnesyltransferase inhibitors, tyrosine kinase inhibitors for exampleinhibitors of the epidermal growth factor family (for example EGFRfamily tyrosine kinase inhibitors such asN-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine(gefitinib, AZD1839),N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine(erlotinib, OSI-774) and6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazolin-4-amine(CI 1033)) and serine/threonine kinase inhibitors); and(iii) antiproliferative/antineoplastic drugs and combinations thereof,as used in medical oncology, such as antimetabolites (for exampleantifolates like methotrexate, fluoropyrimidines like 5-fluorouracil,tegafur, purine and adenosine analogues, cytosine arabinoside);antitumour antibiotics (for example anthracyclines like adriamycin,bleomycin, doxorubicin, daunomycin, epirubicin and idarubicin,mitomycin-C, dactinomycin, mithramycin); platinum derivatives (forexample cisplatin, carboplatin); alkylating agents (for example nitrogenmustard, melphalan, chlorambucil, busulphan, cyclophosphamide,ifosfamide, nitrosoureas, thiotepa); antimitotic agents (for examplevinca alkaloids like vincristine, vinblastine, vindesine, vinorelbine,and taxoids like taxol, taxotere); topoisomerase inhibitors (for exampleepipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan,camptothecin and also irinotecan); also enzymes (for exampleasparaginase); and thymidylate synthase inhibitors (for exampleraltitrexed); and additional types of chemotherapeutic agent include:(iv) biological response modifiers (for example interferon);(v) antibodies (for example edrecolomab);(vi) antisense therapies, for example those which are directed to thetargets listed above, such as ISIS 2503, an anti-ras antisense;(vii) gene therapy approaches, including for example approaches toreplace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2,GDEPT (gene-directed enzyme pro-drug therapy) approaches such as thoseusing cytosine deaminase, thymidine kinase or a bacterial nitroreductaseenzyme and approaches to increase patient tolerance to chemotherapy orradiotherapy such as multi-drug resistance gene therapy; and(viii) immunotherapy approaches, including for example ex-vivo and invivo approaches to increase the immunogenicity of patient tumour cells,such as transfection with cytokines such as interleukin 2, interleukin 4or granulocyte-macrophage colony stimulating factor, approaches todecrease T-cell anergy, approaches using transfected immune cells suchas cytokine-transfected dendritic cells, approaches usingcytokine-transfected tumour cell lines and approaches usinganti-idiotypic antibodies.

For example such conjoint treatment may be achieved by way of thesimultaneous, sequential or separate administration of an AZD2171maleate salt as defined hereinbefore and a vascular targeting agentdescribed in WO 99/02166 such as N-acetylcolchinol-O-phosphate (Example1 of WO 99/02166).

It is known from WO 01/74360 that antiangiogenics can be combined withantihypertensives. A salt of the present invention can also beadministered in combination with an antihypertensive. Anantihypertensive is an agent which lowers blood pressure, see WO01/74360 which is incorporated herein by reference.

Thus according to the present invention there is provided a method oftreatment of a disease state associated with angiogenesis whichcomprises the administration of an effective amount of a combination ofan AZD2171 maleate salt as defined hereinbefore and an anti-hypertensiveagent to a warm-blooded animal, such as a human being.

According to a further feature of the present invention there isprovided the use of a combination of an AZD2171 maleate salt as definedhereinbefore and an anti-hypertensive agent for use in the manufactureof a medicament for the treatment of a disease state associated withangiogenesis in a warm-blooded mammal, such as a human being.

According to a further feature of the present invention there isprovided a pharmaceutical composition comprising an AZD2171 maleate saltas defined hereinbefore and an anti-hypertensive agent for the treatmentof a disease state associated with angiogenesis in a warm-bloodedmammal, such as a human being.

According to a further aspect of the present invention there is provideda method for producing an anti-angiogenic and/or vascular permeabilityreducing effect in a warm-blooded animal, such as a human being, whichcomprises administering to said animal an effective amount of an AZD2171maleate salt as defined hereinbefore and an anti-hypertensive agent.

According to a further aspect of the present invention there is providedthe use of a combination of an AZD2171 maleate salt as definedhereinbefore and an anti-hypertensive agent for the manufacture of amedicament for producing an anti-angiogenic and/or vascular permeabilityreducing effect in a warm-blooded mammal, such as a human being.

Preferred antihypertensive agents are calcium channel blockers,angiotensin converting enzyme inhibitors (ACE inhibitors), angiotensinII receptor antagonists (A-II antagonists), diuretics, beta-adrenergicreceptor blockers (β-blockers), vasodilators and alpha-adrenergicreceptor blockers (α-blockers). Particular antihypertensive agents arecalcium channel blockers, angiotensin converting enzyme inhibitors (ACEinhibitors), angiotensin II receptor antagonists (A-II antagonists) andbeta-adrenergic receptor blockers (β-blockers), especially calciumchannel blockers.

As stated above AZD2171 maleate salt is of interest for itsantiangiogenic and/or vascular permeability reducing effects. AZD2171maleate salt is expected to be useful in a wide range of disease statesincluding cancer, diabetes, psoriasis, rheumatoid arthritis, Kaposi'ssarcoma, haemangioma, lymphoedema, acute and chronic nephropathies,atheroma, arterial restenosis, autoimmune diseases, acute inflammation,excessive scar formation and adhesions, endometriosis, dysfunctionaluterine bleeding and ocular diseases with retinal vessel proliferationincluding age-related macular degeneration. Cancer may affect any tissueand includes leukaemia, multiple myeloma and lymphoma. In particularsuch compounds of the invention are expected to slow advantageously thegrowth of primary and recurrent solid tumours of, for example, thecolon, breast, prostate, lungs and skin. More particularly suchcompounds of the invention are expected to inhibit any form of cancerassociated with VEGF including leukaemia, multiple myeloma and lymphomaand also, for example, the growth of those primary and recurrent solidtumours which are associated with VEGF, especially those tumours whichare significantly dependent on VEGF for their growth and spread,including for example, certain tumours of the colon, breast, prostate,lung, brain vulva and skin.

In addition to their use in therapeutic medicine, the AZD2171 maleatesalts defined hereinbefore are also useful as pharmacological tools inthe development and standardisation of in vitro and in vivo test systemsfor the evaluation of the effects of inhibitors of VEGF receptortyrosine kinase activity in laboratory animals such as cats, dogs,rabbits, monkeys, rats and mice, as part of the search for newtherapeutic agents.

The assays written up in WO 00/47212 and used to test AZD2171 are asfollows:

(a) In Vitro Receptor Tyrosine Kinase Inhibition Test

This assay determines the ability of a test compound to inhibit tyrosinekinase activity. DNA encoding VEGF, FGF or EGF receptor cytoplasmicdomains may be obtained by total gene synthesis (Edwards M,International Biotechnology Lab 5(3), 19-25, 1987) or by cloning. Thesemay then be expressed in a suitable expression system to obtainpolypeptide with tyrosine kinase activity. For example VEGF, FGF and EGFreceptor cytoplasmic domains, which were obtained by expression ofrecombinant protein in insect cells, were found to display intrinsictyrosine kinase activity. In the case of the VEGF receptor Flt-1(Genbank accession number X51602), a 1.7 kb DNA fragment encoding mostof the cytoplasmic domain, commencing with methionine 783 and includingthe termination codon, described by Shibuya et al (Oncogene, 1990, 5:519-524), was isolated from cDNA and cloned into a baculovirustransplacement vector (for example pAcYM1 (see The BaculovirusExpression System: A Laboratory Guide, L. A. King and R. D. Possee,Chapman and Hall, 1992) or pAc360 or pBlueBacHis (available fromInvitrogen Corporation)). This recombinant construct was co-transfectedinto insect cells (for example Spodoptera frugiperda 21(Sf21)) withviral DNA (eg Pharmingen BaculoGold) to prepare recombinant baculovirus.(Details of the methods for the assembly of recombinant DNA moleculesand the preparation and use of recombinant baculovirus can be found instandard texts for example Sambrook et al, 1989, Molecular cloning—ALaboratory Manual, 2nd edition, Cold Spring Harbour Laboratory Press andO'Reilly et al, 1992, Baculovirus Expression Vectors—A LaboratoryManual, W. H. Freeman and Co, New York). For KDR (Genbank accessionnumber L04947), a cytoplasmic fragment starting from methionine 806 wascloned and expressed in a similar manner.

For expression of cFlt-1 tyrosine kinase activity, Sf21 cells wereinfected with plaque-pure cFlt-1 recombinant virus at a multiplicity ofinfection of 3 and harvested 48 hours later. Harvested cells were washedwith ice cold phosphate buffered saline solution (PBS) (10 mM sodiumphosphate pH7.4, 138 mM sodium chloride, 2.7 mM potassium chloride) thenresuspended in ice cold HNTG/PMSF (20 mM Hepes pH7.5, 150 mM sodiumchloride, 10% v/v glycerol, 1% v/v Triton X100, 1.5 mM magnesiumchloride, 1 mM ethylene glycol-bis(βaminoethyl ether)N,N,N′,N′-tetraacetic acid (EGTA), 1 mM PMSF (phenylmethylsulphonylfluoride); the PMSF is added just before use from a freshly-prepared 100mM solution in methanol) using 1 ml HNTG/PMSF per 10 million cells. Thesuspension was centrifuged for 10 minutes at 13,000 rpm at 4° C., thesupernatant (enzyme stock) was removed and stored in aliquots at −70° C.Each new batch of stock enzyme was titrated in the assay by dilutionwith enzyme diluent (100 mM Hepes pH 7.4, 0.2 mM sodium orthovanadate,0.1% v/v Triton X100, 0.2 mM dithiothreitol). For a typical batch, stockenzyme is diluted 1 in 2000 with enzyme diluent and 50 μl of diluteenzyme is used for each assay well.

A stock of substrate solution was prepared from a random copolymercontaining tyrosine, for example Poly (Glu, Ala, Tyr) 6:3:1 (SigmaP3899), stored as 1 mg/ml stock in PBS at −20° C. and diluted 1 in 500with PBS for plate coating.

On the day before the assay 100 μl of diluted substrate solution wasdispensed into all wells of assay plates (Nunc maxisorp 96-wellimmunoplates) which were sealed and left overnight at 4° C.

On the day of the assay the substrate solution was discarded and theassay plate wells were washed once with PBST (PBS containing 0.05% v/vTween 20) and once with 50 mM Hepes pH7.4.

Test compounds were diluted with 10% dimethylsulphoxide (DMSO) and 25 μlof diluted compound was transferred to wells in the washed assay plates.“Total” control wells contained 10% DMSO instead of compound. Twentyfive microlitres of 40 mM manganese(II)chloride containing 8 μMadenosine-5′-triphosphate (ATP) was added to all test wells except“blank” control wells which contained manganese(II)chloride without ATP.To start the reactions 50 μl of freshly diluted enzyme was added to eachwell and the plates were incubated at room temperature for 20 minutes.The liquid was then discarded and the wells were washed twice with PBST.One hundred microlitres of mouse IgG anti-phosphotyrosine antibody(Upstate Biotechnology Inc. product 05-321), diluted 1 in 6000 with PBSTcontaining 0.5% w/v bovine serum albumin (BSA), was added to each welland the plates were incubated for 1 hour at room temperature beforediscarding the liquid and washing the wells twice with PBST. One hundredmicrolitres of horse radish peroxidase (HRP)-linked sheep anti-mouse Igantibody (Amersham product NXA 931), diluted 1 in 500 with PBSTcontaining 0.5% w/v BSA, was added and the plates were incubated for 1hour at room temperature before discarding the liquid and washing thewells twice with PBST. One hundred microlitres of2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) solution,freshly prepared using one 50 mg ABTS tablet (Boehringer 1204 521) in 50ml freshly prepared 50 mM phosphate-citrate buffer pH5.0+0.03% sodiumperborate (made with 1 phosphate citrate buffer with sodium perborate(PCSB) capsule (Sigma P4922) per 100 ml distilled water), was added toeach well. Plates were then incubated for 20-60 minutes at roomtemperature until the optical density value of the “total” controlwells, measured at 405 nm using a plate reading spectrophotometer, wasapproximately 1.0. “Blank” (no ATP) and “total” (no compound) controlvalues were used to determine the dilution range of test compound whichgave 50% inhibition of enzyme activity.

(b) In Vitro HUVEC Proliferation Assay

This assay determines the ability of a test compound to inhibit thegrowth factor-stimulated proliferation of human umbilical veinendothelial cells (HUVEC).

HUVEC cells were isolated in MCDB 131 (Gibco BRL)+7.5% v/v foetal calfserum (FCS) and were plated out (at passage 2 to 8), in MCDB 131+2% v/vFCS+3 μg/ml heparin+1 μg/ml hydrocortisone, at a concentration of 1000cells/well in 96 well plates. After a minimum of 4 hours they were dosedwith VEGF (3 ng/ml) and compound. The cultures were then incubated for 4days at 37° C. with 7.5% CO₂. On day 4 the cultures were pulsed with 1μCi/well of tritiated-thymidine (Amersham product TRA 61) and incubatedfor 4 hours. The cells were harvested using a 96-well plate harvester(Tomtek) and then assayed for incorporation of tritium with a Beta platecounter. Incorporation of radioactivity into cells, expressed as cpm,was used to measure inhibition of growth factor-stimulated cellproliferation by compounds. This methodology was also used to assesscompound effects versus basal HUVEC growth (i.e. endothelial cellproliferation in MCDB 131+2% v/v FCS+3 μg/ml heparin+1 μg/mlhydrocortisone without the addition of exogenous VEGF).

(c) In Vivo Solid Tumour Disease Model

This test measures the capacity of compounds to inhibit solid tumourgrowth.

CaLu-6 tumour xenografts were established in the flank of female athymicSwiss nu/nu mice, by subcutaneous injection of 1×10⁶ Calu-6 cells/mousein 100 μl of a 50% (v/v) solution of Matrigel in serum free culturemedium. Ten days after cellular implant, mice were allocated to groupsof 8-10, so as to achieve comparable group mean volumes. Tumours weremeasured using vernier calipers and volumes were calculated as:(l×w)×√(l×w)×(π/6), where l is the longest diameter and w the diameterperpendicular to the longest. Test compounds were administered orallyonce daily for a minimum of 21 days, and control animals receivedcompound diluent. Tumours were measured twice weekly. The level ofgrowth inhibition was calculated by comparison of the mean tumour volumeof the control group versus the treatment group using a Student T testand/or a Mann-Whitney Rank Sum Test. The inhibitory effect of compoundtreatment was considered significant when p<0.05.

An AZD2171 maleate salt as defined hereinbefore may be prepared by anyprocess known to be applicable to the preparation of chemically-relatedcompounds. Such processes include, for example, those illustrated inInternational Patent Application No. WO 00/47212 all of which areincorporated herein by reference. Such processes also include, forexample, solid phase synthesis. Such processes, are provided as afurther feature of the invention and are as described hereinafter.Necessary starting materials may be obtained by standard procedures oforganic chemistry. AZD2171 free base may be prepared according to any ofthe processes described in WO 00/47212, see in particular Example 240 ofWO 00/47212. Alternatively necessary starting materials are obtainableby analogous procedures to those illustrated which are within theordinary skill of an organic chemist.

The following processes (a) (b) and (c) constitute further features ofthe present invention.

Synthesis of AZD2171 Maleate Salt Form A

(a) Such a process provides a further aspect of the present inventionand comprises, for example, the steps of:

-   -   (i) dissolving AZD2171 free base in an organic solvent to form a        solution;    -   (ii) adding an aqueous solution of maleic acid or adding a        solution of maleic acid in an organic solvent;    -   (iii) allowing spontaneous nucleation to occur;    -   (iv) optionally isolating the crystalline mixture of AZD2171        Forms A and B so formed;    -   (v) slurrying the mixture in a solvent, for example methanol,        until all the AZD2171 maleate is Form A, (as may be determined        by X-Ray Powder Diffraction), for example this may take 4 days;        and    -   (vi) isolating the crystalline solid so formed.        (b) Another such process provides a further aspect of the        present invention and comprises, for example, the steps of:    -   (i) dissolving AZD2171 free base in an organic solvent to form a        solution;    -   (ii) adding an aqueous solution of maleic acid or adding a        solution of maleic acid in an organic solvent;    -   (iii) obtaining a solution, for example by heating or adding        more solvent, and adding a seed of AZD2171 maleate Form A to        initiate crystallisation of AZD2171 maleate Form A; and    -   (iv) isolating the crystalline solid so formed.

For part (i) of (a) and (b) the mixture may, if required, be heated toreflux until dissolution has occurred. Alternatively, the mixture may,for example, be heated to a temperature less than the reflux temperatureof the solvent provided that dissolution of more or less all of thesolid material has occurred. It will be appreciated that smallquantities of insoluble material may be removed by filtration of thewarmed mixture.

For part (i) of (a) and (b) the organic solvent is preferably analcohol, for example methanol or isopropanol.

For part (ii) of (a) and (b) the organic solvent is preferably analcohol, for example methanol.

(c) Synthesis of AZD2171 Maleate Salt Form B

Such a process provides a further aspect of the present invention andcomprises, for example, the steps of:

-   -   (i) dissolving AZD2171 maleate in an organic solvent to form a        solution;    -   (ii) adding the solution to a solvent in which AZD2171 maleate        has a lower solubility than it does in NMP, for example toluene        or ethyl acetate;    -   (iii) crystallisation of AZD2171 maleate Form B then occurs; and    -   (iv) isolating the crystalline solid so formed.        -   In (c) a preferred organic solvent is a highly solubilising            solvent such as 1-methyl-2-pyrrolidinone.

For part (i) of (c) the mixture may, if required, be heated to refluxuntil dissolution has occurred. Alternatively, the mixture may, forexample, be heated to a temperature less than the reflux temperature ofthe solvent provided that dissolution of more or less all of the solidmaterial has occurred. It will be appreciated that small quantities ofinsoluble material may be removed by filtration of the warmed mixture.

In (a), (b) and (c) above the crystalline solid so formed may beisolated by any conventional method, for example by filtration.

The invention is illustrated hereinafter by means of the followingnon-limiting Examples, data and Figures in which, unless otherwisestated:—

(i) evaporations were carried out by rotary evaporation in vacuo andwork-up procedures were carried out after removal of residual solidssuch as drying agents by filtration;

(ii) yields are given for illustration only and are not necessarily themaximum attainable;

(iii) melting points are uncorrected and were determined using a MettlerDSC820e; (iv) the structures of the end-products of the formula I wereconfirmed by nuclear (generally proton) magnetic resonance (NMR) andmass spectral techniques; proton magnetic resonance chemical shiftvalues were measured on the delta scale and peak multiplicities areshown as follows: s, singlet; d, doublet; t, triplet; m, multiplet; br,broad; q, quartet, quin, quintet; all samples run on a Bruker DPX 400MHz at 300K in d₆-DMSO, 16 scans, pulse repetition time 10 seconds;

(v) intermediates were not generally fully characterised and purity wasassessed by NMR analysis; and

(vi) the following abbreviations have been used:—

-   -   DMSO dimethylsulphoxide    -   NMP 1-methyl-2-pyrrolidinone

EXAMPLE 1: AZD2171 MALEATE FORM A

Under an inert atmosphere of nitrogen AZD2171 crude free base (4.52 g),(prepared for example as described in Example 240 of WO 00/47212) wasslurried with isopropanol (58.8 mL). The mixture was heated at refluxfor 15 minutes to give a clear, dark solution. The mixture was cooled to75° C. and charcoal (0.226 g) added. The mixture was reheated to refluxand held at reflux for an hour. The mixture was then filtered hot. Thecharcoal filter cake was washed with hot isopropanol (9 mL). Thetemperature of the combined filtrate and wash was adjusted to 55° C. anda prefiltered solution of maleic acid (1.173 g) in water (2.71 mL) wasadded dropwise over 5 minutes. The crude free base which previouslycrystallised dissolved during the addition. A line wash of water (0.9mL) was added. The mixture was maintained at 55° C. for 15 minutes and aseed of AZD2171 maleate Form A (0.023 g) added. The mixture was held at55° C. for 4 hours. During the 4 hour hold crystallisation becameestablished. The mixture was cooled to 0° C. over 8 hours. The mixturewas held at 0° C. for a minimum of 8 hours. The mixture was filtered.The cake was washed with isopropanol (9 mL). The solid was dried in avacuum oven at 50° C. to give4-([4-fluoro-2-methyl-1H-indol-5-yl]oxy)-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazolinemaleate Form A. ¹H NMR Spectrum: (400 MHz, DMSO): 11.36 (s, 1H), 8.53(s, 1H), 7.65 (s, 1H), 7.43 (s, 1H), 7.18 (d, 1H), 7.01 (d, 1H), 6.25(s, 1H), 6.04 (s, 2H), 4.33 (t, 2H), 4.02 (s, 3H), 3.26-3.3.70 (b, 4H),2.44, (s, 3H), 2.24 (m, 2H), 2.02 (m, 4H).

m.p.: DSC analysis: onset of melting at 198.3° C. and a peak at 200.08°C.

EXAMPLE 2: AZD2171 MALEATE FORM A

Under an inert atmosphere of nitrogen AZD2171 crude free base (23.0 g)(prepared for example as described in Example 240 of WO 00/47212) wasslurried in methanol (223 mL) in vessel 1. The mixture was degassed byholding under vacuum and then releasing the vacuum with nitrogen. Thiswas repeated five times. The slurry was then heated to reflux and heldthere for 15 minutes to give a clear, dark brown solution. The solutionwas cooled to 60° C. and then filtered through a Celite® pad (4.00 g)into vessel 2. The Celite® pad was washed with hot (60° C.) methanol (78mL), the filtrate again going to vessel 2.

To vessel 1 was then charged methanol (111 mL), which was cooled to 0°C. To vessel 1 was then charged maleic acid (5.50 g) and the mixturestirred at 0° C. for 15 minutes until all the maleic acid had dissolved.

The contents of vessel 1 were then charged to vessel 2 through anin-line filter whilst maintaining the temperature above 52° C. A seed ofAZD2171 maleate Form A (0.0454 g) was added to vessel 2 at 55° C. andthe mixture held at 55° C. for 3 hours. The mixture was then cooled to40° C. over 7 hours, then cooled further to −5° C. over 6 hours. Thesolid was filtered and washed with methanol (100 mL) at −5° C. Theproduct was dried in a vacuum oven for 24 hours to give4-([4-fluoro-2-methyl-1H-indol-5-yl]oxy)-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazolinemaleate Form A.

EXAMPLE 3: AZD2171 MALEATE FORM B

AZD2171 maleate Form A (2.31 g) was dissolved in warm (˜50° C.) NMP.This solution was added dropwise to toluene (23 mL) over 2 minutes atambient temperature. Material originally precipitated as a solid thenbecame an oil, then a solid again. After stirring for 10 minutes atambient temperature the solid was filtered and washed with toluene (10mL). The solid was dried in a vacuum oven at ambient temperatureovernight to give4-((4-fluoro-2-methyl-1H-indol-5-yl)oxy)-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazolinemaleate Form B.

m.p.: DSC analysis: onset of melting at 194.43° C. and a peak at 195.97°C.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: DSC and TGA Thermograms for AZD2171 Free base Monohydrate—withtemperature in ° C. plotted on the horizontal axis and heat flow/%weight loss on the vertical axis

FIG. 2: X-Ray Powder Diffraction Pattern for AZD2171 free base—with the2θ values plotted on the horizontal axis and the relative line intensity(count) plotted on the vertical axis.

FIG. 3: X-Ray Powder Diffraction Pattern for AZD2171 Free baseMonohydrate Heated to 100° C.—with the 2θ values plotted on thehorizontal axis and the relative line intensity (count) plotted on thevertical axis.

FIG. 4: X-Ray Powder Diffraction Pattern for AZD2171 Free baseMicronised—with the 20 values plotted on the horizontal axis and therelative line intensity (count) plotted on the vertical axis.

FIG. 5: X-Ray Powder Diffraction Pattern for AZD2171 Maleate Salt FormA—with the 20 values plotted on the horizontal axis and the relativeline intensity (count) plotted on the vertical axis.

FIG. 6: DSC Thermogram for AZD2171 Maleate Form A—with temperature in °C. plotted on the horizontal axis and endothermic heat flow (milliWatts(mW)) plotted on the vertical axis.

FIG. 7: AZD2171 Maleate Form A Vapour Sorption Isotherm at 25° C.—withtarget relative humidity (RH) (%) plotted on the horizontal axis andchange in dry mass (%) plotted on the vertical axis.

FIG. 8: X-Ray Powder Diffraction Pattern AZD2171 Maleate Salt FormB—with the 20 values plotted on the horizontal axis and the relativeline intensity (count) plotted on the vertical axis.

FIG. 9: DSC Thermogram for AZD2171 Maleate Form B—with temperature in °C. plotted on the horizontal axis and endothermic heat flow (milliWatts(mW)) plotted on the vertical axis.

FIG. 10: X-Ray Powder Diffraction Patterns for AZD2171 Maleate SlurryExperiment with the 2θ values plotted on the horizontal axis and therelative line intensity (count) plotted on the vertical axis.

DETAILS OF TECHNIQUES USED X-Ray Powder Diffraction

TABLE 5 % Relative Intensity* Definition  25-100 vs (very strong) 10-25s (strong)  3-10 m (medium) 1-3 w (weak) *The relative intensities arederived from diffractograms measured with fixed slits

Analytical Instrument: Siemens D5000

The X-ray powder diffraction spectra were determined by mounting asample of the crystalline salt on Siemens single silicon crystal (SSC)wafer mounts and spreading out the sample into a thin layer with the aidof a microscope slide. The sample was spun at 30 revolutions per minute(to improve counting statistics) and irradiated with X-rays generated bya copper long-fine focus tube operated at 40 kV and 40 mA with awavelength of 1.5406 angstroms. The collimated X-ray source was passedthrough an automatic variable divergence slit set at V20 and thereflected radiation directed through a 2 mm antiscatter slit and a 0.2mm detector slit. The sample was exposed for 1 second per 0.02 degree2-theta increment (continuous scan mode) over the range 2 degrees to 40degrees 2-theta in theta-theta mode. The running time was 31 minutes and41 seconds. The instrument was equipped with a scintillation counter asdetector. Control and data capture was by means of a Dell Optiplex 686NT 4.0 Workstation operating with Diffract+ software. Persons skilled inthe art of X-ray powder diffraction will realise that the relativeintensity of peaks can be affected by, for example, grains above 30microns in size and non-unitary aspect ratios which may affect analysisof samples. The skilled person will also realise that the position ofreflections can be affected by the precise height at which the samplesits in the diffractometer and the zero calibration of thediffractometer. The surface planarity of the sample may also have asmall effect. Hence the diffraction pattern data presented are not to betaken as absolute values.

Sieving/Micronisation

AZD2171 free base was sieved prior to Micronising using a 1 mm stainlesssteel sieve, the base being used for product collection and for manualfeeding directly into the microniser. Approximately 7.5 g of AZD2171free base was sieved.

A clean S/S lined 2″ Microniser was used.

Manual feed rate: approximately 2/3 g per minute.

Grind air pressure range 10/20 psi (0.67/1.33 atmospheres).

Venturi air pressure range 20/25 psi (1.33/1.67 atmospheres).

Dynamic Vapour Sorption

Analytical Instrument: Surface Measurements Systems Dynamic VapourSorption Analyser.

About 5 mg of material contained in a quartz holder at 25° C. wassubjected to humidified nitrogen at the following relative humidities(RH): 0, 20, 40, 60, 80, 95, 80, 60, 40, 20, 0% RH in duplicate.

Differential Scanning Calorimetry

Analytical Instrument: Mettler DSC820e.

Typically less than 5 mg of material contained in a 40 μl aluminium panfitted with a pierced lid was heated over the temperature range 25° C.to 325° C. at a constant heating rate of 10° C. per minute. A purge gasusing nitrogen was used—flow rate 100 ml per minute.

Thermogravimetric Analysis

Analytical Instrument: Mettler TG851.

Typically between 3 and 12 mg of material contained in a 70 μl alox(aluminium oxide) crucible was heated over the temperature range 25° C.to 325° C. at a constant heating rate of 10° C. per minute. A purge gasusing helium was used—flow rate 50 ml per minute.

Karl Fischer Water Content

Analytical Instrument: Mitsubishi Moisture Meter CA-05.

Typically approximately 50 mg of material was used.

1-20. (canceled)
 21. A method for inhibiting VEGF receptor tyrosinekinase in a warm-blooded animal in need of such treatment whichcomprises orally administering to said animal an effective amount of apharmaceutical composition comprising crystalline Form A of4-((4-fluoro-2-methyl-1H-indol-5-yl)oxy)-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazolinemaleate, wherein said warm-blooded animal in need of such treatment hasa solid tumor cancer that is recurrent.
 22. The method according toclaim 21, wherein said cancer is a female reproductive cancer.
 23. Themethod according to claim 21, wherein the daily dose of Form A of4-((4-fluoro-2-methyl-1H-indol-5-yl)oxy)-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazolinemaleate administered to said animal ranges from 0.03 mg/kg to 0.5 mg/kg.24. The method according to claim 21, further comprising administeringat least one additional treatment chosen from surgery, radiotherapy, andchemotherapy.
 25. The method according to claim 24, wherein saidchemotherapy comprises at least one therapeutic agent chosen from (i)other antiangiogenic agents; (ii) cytostatic agents; and (iii)antiproliferative/antineoplastic drugs.
 26. The method according toclaim 25, wherein said antiproliferative/antineoplastic drugs are chosenfrom platinum derivatives.